Productions of hetero-oligomeric proteins in plants

ABSTRACT

Process of producing in a plant, in plant tissue, or in plant cells a hetero-oligomeric protein comprising at least a first and a second protein subunit, said process comprising expressing in plant cells at least said first and said second protein subunit by
     (i) providing to said plant, said plant tissue or said plant cells a plus-sense single-stranded RNA viral vector encoding at least said first and said second protein subunit or   (ii) providing to said plant, said plant tissue or said plant cells a first and a second plus-sense single-stranded RNA viral vector, said first viral vector encoding at least said first protein subunit, said second viral vector encoding at least said second protein subunit, whereby at least said first viral vector and said second viral vector are non-competing viral vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/814,931, filed Jul. 27, 2007, which is the U.S. National Stage ofInternational Application PCT/EP2006/000721 filed Jan. 27, 2006, whichclaims the benefit of U.S. Provisional Application No. 60/593,606 filedJan. 28, 2005 and EP 05001819.1 filed Jan. 28, 2005; all of which arehereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to the production of hetero-oligomericproteins in plant, plant parts or plant cell cultures using theplus-sense single stranded RNA viral vectors. The process and vectorsdescribed in the present invention provide plant cells with an increasedyield of functional hetero-oligomeric recombinant protein, such asfull-length antibody or hetero-oligomeric synthetic derivatives thereofincluding fusions with other proteins or their fragments.

BACKGROUND OF THE INVENTION

Plant-based molecular farming is an attractive opportunity for theproduction of recombinant proteins destined to be used in the field ofhuman and animal health, preferably due to the potentially lowproduction cost and attempts by biopharmaceutical industry to eliminateanimal-derived proteins from manufacturing processes because of possiblecontamination of these products by human pathogens such as BovineSpongiform Encephalopathy (BSE) or Creuzfeld-Jacob Disease (CJD, vCJD).However, high-yield production of hetero-oligomeric proteins in plantcells is a problem that cannot be resolved with the help of standardexpression systems based on the use of strong constitutive ortissue-specific promoters for the following reasons: firstly, themajority of such recombinant proteins have a deleterious effect on plantgrowth and development, thus strongly compromising the yield; secondly,use of tissue-specific promoters (e.g. seed-specific) would requirestable incorporation of genes encoding for pharmaceutical proteins intothe genomes of edible crop plants (e.g. rice, corn, wheat), that mightcause problems with transgene flow control in case of open fieldcultivation. Also, these systems are not commercially viable, if used inclosed (greenhouse) environment due to the low yield of the product.

Plant virus-based transient expression systems (for review see: Porta &Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999,Curr. Top. Microbiol. Immunol., 240, 81-94; Gleba et al., 2004, Curr.Opin. Plant Biol., 7, 182-188) are able to provide for high expressionlevels in plant leaf tissues and to some extent are capable to addressthe problems of cytotoxicity of recombinant proteins and theirdetrimental effect on plant development, as the technology allows toseparate the growth and production stages. The best-established andcommercially viable systems are based on plus-sense single-stranded RNAviral vectors, preferably on Tobacco Mosaic Virus (TMV)-derived vectors(Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Malloryet al., 2002, Nature Biotechnol. 20, 622-625; U.S. Pat. No. 5,316,931;U.S. Pat. No. 5,589,367; U.S. Pat. No. 5,866,785; U.S. Pat. No.5,977,438; WO02088369; WO02097080; WO0229068; U.S. Pat. No. 5,491,076).However these systems suffer from serious limitations that restricttheir use to the production of simple, relatively small proteins. Inpart this is caused by the instability of viral vectors and the highfrequency of their reversion to wild type, if they carry heterologoussequences larger than 1 kb. Also, serious limitation of the technologyis the absence of viral vector systems capable of expressing complexhetero-oligomeric proteins like therapeutic monoclonal antibodies andtheir derivatives that represent the most valuable group of recombinantproteins.

There is only one publication addressing the expression of a full-lengthmonoclonal antibody in plants using plant viral vectors (Verch et al.,1998, J. Immunol. Meth., 220, 6975). This paper describes the use of twosystemic TMV-based viral vectors for the expression of heavy and lightchains of monoclonal antibody in systemic leaves, whereby the differentchains are expressed from different vectors upon co-infection of N.benthamiana plants with in vitro synthesised transcripts of saidvectors. However, the yield of recombinant protein in said system is solow that the presence of assembled monoclonal antibody had to beconfirmed with highly sensitive tests like Western blotting and ELISA.Due to the negligible yield of recombinant antibody, this system is notsuitable for practical applications and has no commercial value. Sincetwo or more TMV-based viral vectors are normally not present in the sameplant tissues of an infected plant (see example 1), the detected antigenbinding activity may be due to antibody that was in vitro assembledduring the isolation procedure from heavy and light antibody chainsexpressed in separate cells or tissue. It was previously shown thatfunctional antibodies can be assembled in vitro from denatured andreduced antibody components (Petersen & Dorrington, 1974, J. Biol.Chem., 17, 5633-5641; Maeda et al., 1996, Protein Engineering, 9,95-100). However, the efficiency of such assembly in the absence ofconditions favourable for such an assembly is very low.

Therefore, there is no large-scale expression system for recombinanthetero-oligomeric proteins in plants, the yield and efficiency of whichwould be sufficient to compete on the market with other large-scaleexpression systems like fungal or insect cell expression systems. Such aplant expression would have to fulfil the following criteria as good aspossible:

-   (i) high yield, including expression of the hetero-oligomeric    protein of interest in as many plant tissues as possible and in as    many cells of said tissues;-   (ii) for preventing a deleterious effect of recombinant protein    expression on plant growth, expression of the protein or product of    interest should be transient (or switchable) such that expression    can be started at a desired stage of plant development;-   (iii) to provide for an optimal ratio of polyproteins encoding for    different subunits of hetero-oligomeric protein in plant cell, thus    supporting for high yield of recombinant protein at the level of    said recombinant protein assembly from said subunits.

Therefore, it is an object of this invention to provide an efficientprocess of producing a hetero-oligomeric protein in a plant, plant part,or plant cell culture. A further object is the provision of a high-yieldplant expression system capable of expressing hetero-oligomeric protein.It is another object of the invention to provide an efficient process ofco-expressing more than one polypeptide of interest in the same plantcell. Further, it is an object of the invention to provide a fast andhigh-yield method for expressing antibodies in a plant, plant part orplant cell culture.

GENERAL DESCRIPTION OF THE INVENTION

The invention provides a process of producing in a plant, in planttissue, or in plant cells a hetero-oligomeric protein comprising atleast a first and a second protein subunit, said process comprisingexpressing in plant cells at least said first and said second proteinsubunit by

-   (i) providing to said plant, said plant tissue or said plant cells a    first and a second plus-sense single-stranded RNA viral vector, said    first viral vector encoding at least said first protein subunit,    said second viral vector encoding at least said second protein    subunit, whereby at least said first viral vector and said second    viral vector are non-competing viral vectors; or-   (ii) providing to said plant, said plant tissue or said plant cells    a plus-sense single-stranded RNA viral vector encoding at least said    first and said second protein subunit.

In one embodiment, said first viral vector and said second viral vectorare non-competing in that they are different viral vectors. In anotherembodiment, said first viral vector and said second viral vector arenon-competing in that they are not derived from the same RNA virus. Inanother embodiment, said first viral vector and said second viral vectorare non-competing in that they differ in a sequence portion other thansequence portions coding for said first and second protein subunits. Inanother embodiment, said first viral vector and said second viral vectorare non-competing in that they are derived from RNA viruses belonging todifferent virus species. In another embodiment, said first viral vectorand said second viral vector are non-competing in that they are derivedfrom RNA viruses belonging to different virus genera. Another embodimentof the invention is a process of producing in a plant, in plant tissue,or in plant cells an antibody comprising at least a first and a secondprotein subunit like a heavy and a light antibody chain, said processcomprising expressing in plant cells at least said first and said secondprotein subunit by

-   (i) providing to said plant, said plant tissue or said plant cells    by Agrobacterium-mediated transfection a DNA precursor of a first    plus-sense single-stranded RNA viral vector and a DNA precursor of a    second plus-sense single-stranded RNA viral vector, said first viral    vector encoding at least said first protein subunit, said second    viral vector encoding at least said second protein subunit, whereby    at least said first viral vector or said second viral vector lack an    open reading frame coding for a functional protein necessary for    systemic movement of said first or said second viral vector in said    plant; or-   (ii) providing to said plant, said plant tissue or said plant cells    by Agrobacterium-mediated transfection a DNA precursor of a    plus-sense single-stranded RNA viral vector encoding at least said    first and said second protein subunit, whereby said viral vector    lacks an ORF coding for a functional protein necessary for systemic    movement.

The invention further provides a process of producing in a plant, inplant tissue or in plant cells a hetero-oligomeric protein comprising atleast a first and a second protein subunit, said process comprisingexpressing in plant cells at least said first and said second proteinsubunit from one or more plus-sense single-stranded RNA viral vector(s).

Further embodiments of the invention are described in the claims and thefollowing description.

The inventors of the present invention have surprisingly identified waysof producing hetero-oligomeric proteins with high yield in plants usingplant viral vectors. Efficient production of hetero-oligomeric proteinsin plants requires high-yield production of the different proteinsubunits of the hetero-oligomeric protein in the same plant cells. Inthis way, the hetero-oligomeric protein can be efficiently assembled incells having expressed said at least two protein subunits using naturalprotein assembling capabilities of said cells like those of theendoplasmatic reticulum (ER). Inefficient in vitro assembly of saidhetero-oligomeric protein is therefore not necessary. The presentinvention achieves for the first time efficient co-expression of two ormore proteins in the same cells by the above step (i) or by the abovestep (ii) or by a combination of the above steps (i) and (ii).

Said first protein subunit is encoded in the RNA viral vector by a firstheterologous (nucleic acid) sequence. Said second protein subunit isencoded in the viral vector by a second heterologous (nucleic acid)sequence. These heterologous sequences are thus RNA sequences of saidviral vector(s) and typically comprise or encode regulatory sequencesrequired for expressing said protein subunits. Examples of suchregulatory sequences are subgenomic promoters, IRES elements, and3′-untranslated sequences. Herein, a sequence is a heterologous sequenceif it does not naturally occur in the virus from which said viralvector(s) is/are derived.

The hetero-oligomeric protein producible according to the presentinvention has at least a first and a second subunit, whereby said firstand said second subunits have different polypeptide sequences. Thus,said first and said second subunit typically have to be expressed fromdifferent heterologous nucleic acid sequences. The subunits of anoligomeric protein assemble to form the quaternery structure of theoligomeric protein. Assembly of the subunits typically involvesnon-covalent bonds between the subunits. Additionally, covalent bondsmay be formed between said protein subunits, e.g. disulfide bonds.

In one embodiment, said hetero-oligomeric protein is a hetero-dimericprotein, i.e. a protein having two different protein subunits. Inanother embodiment, said hetero-oligomeric protein may have more thantwo different subunits e.g. 3 or 4 different subunits (hetero-trimericor hetero-tetrameric protein, respectively). In a further embodiment,said hetero-oligomeric protein may have two different subunits, wherebyone or both of said subunits may be present in said hetero-oligomericprotein more than one time. Examples of the subunit organization of saidhetero-oligomeric protein are AaBb, AaBbCc, and AaBbCcDd, wherein Astands for a first protein subunit, B stand for a second proteinsubunit, and C and D stand for further protein subunits. Each capitalletter A, B, C, and D stands for protein subunits different from theother protein subunits and small letters stand for integers of at least1 that indicate the number of copies of the respective protein subunitin said hetero-oligomeric protein. An example are IgG antibodies whichhave the subunit organization A2B2, wherein A represents a first proteinsubunit (e.g. the heavy chain) and B represents a second protein subunit(e.g. the light chain). Preferably, the hetero-oligomeric proteinproduced according to the invention has two or three different proteinsubunits, more preferably it has two different protein subunits.

In the process of the invention, at least said first and said secondprotein subunits are expressed in cells of said plant, of said planttissue, or of said plant cells by said step (i) or/and said step (ii).Each of said steps (i) and (ii) allows expression of at least said firstand said second protein subunit in the same cells such that saidhetero-oligomeric protein can be produced efficiently in said cells.Steps (i) and (ii) can be performed in parallel, notably for theproduction of hetero-oligomeric proteins having three, four, or moredifferent protein subunits (see example 7).

Said plus-sense single-stranded RNA viral vector(s) of the invention arealso referred to herein simply as “viral vector”. Typically, saidplus-sense single-stranded RNA viral vectors are derived from plus-sensesingle-stranded plant RNA viruses.

In step (ii), said plant, said plant tissue or said plant cells is/areprovided with a plus-sense single-stranded RNA viral vector encoding atleast said first and said second protein subunit. Said viral vectorencoding at least said first and said second protein subunit contains,as an insert, a first heterologous sequence encoding said first proteinsubunit expression of which may be under the control of a firstsub-genomic promoter. Further, said viral vector contains, as an insert,a second heterologous sequence encoding said second protein subunitexpression of which may be under the control of a second sub-genomicpromoter. If both said first and said second protein subunits areexpressed under the control of a subgenomic promoter, these subgenomicpromoters preferably differ in sequence for avoiding self-homology insaid viral vector, which could lead to undesired recombination events inplant cells. Such different subgenomic promoters may be taken fromdifferent strains or species of a plant virus, e.g. one subgenomicpromoter may be (or may be derived from) the coat protein (CP)subgenomic promoter of tobacco mosaic virus (TMV) U1 and the othersubgenomic promoter may be (or may be derived from) the CP subgenomicpromoter of TMV U5 or from crucifer-infecting tobamovirus (cr-TMV).

Instead of said first or said second subgenomic promoter, translation ofsaid first or said second protein subunit may be under control of anIRES (internal ribosome entry site) element. Although translation ofboth said first and said second protein subunits may be under thecontrol of IRES elements, it is preferred that at least one of saidprotein subunits is expressed using a subgenomic promoter. The IRESelements for use in the present invention may be taken from plantviruses like cr-TMV or other plant viruses (Proc Natl Acad Sci USA 2002,99, 5301-6; Virology 1999, 263, 139-54; WO03020927; WO0229068).

Said viral vector of step (ii) is preferably incapable of systemicmovement in said plant or said plant tissue. This can be achieved e.g.by omitting a functional origin of viral particle assembly. Intobamoviruses for example, the origin of viral particle assembly islocated in the MP ORF. Thus the origin of viral particle assembly can beomitted by deleting fully or partly the MP ORF from said viral vector.Said viral vector is thus preferably devoid of a functional movementprotein (MP) ORF. More preferably, said viral vector is devoid of afunctional protein necessary for systemic movement of said viral vector.In this embodiment, said viral vector may be devoid of a functional coatprotein ORF, and most preferably said viral vector is devoid of both afunctional movement protein ORF and a functional coat protein ORF.Omitting an MP ORF and/or a CP ORF from the viral vector provides morespace for encoding at least said first and said second protein subunitin said viral vector without compromising viral vector stability. Inthis embodiment, said viral vector is preferably provided to many cellsof said plant or said plant tissue for achieving infection of manycells. This may best be achieved by providing a DNA precursor of saidRNA viral vector as T-DNA using Agrobacterium (see below).

The virus said viral vector for step (ii) is derived from may be anyplus-sense single-stranded plant RNA virus, e.g. those listed in chapter“Detailed Description”. Preferred groups of viruses are tobamoviruses,potexviruses, and potyviruses. Most preferred viruses are TMV and PVX.Said viral vector will at least contain the ORFs from the virus it isderived from that encode proteins required for replication of said viralvector. Said viral vector typically further contains regulatory elementsfor viral replication and at least one subgenomic promoter.

In step (i), said plant, said plant tissue or said plant cells areprovided with a first and a second plus-sense single-stranded RNA viralvector. Said first viral vector encodes at least said first proteinsubunit, said second viral vector encodes at least said second proteinsubunit.

Step (i) allows to produce hetero-oligomeric proteins having twodifferent protein subunits. Step (i) also allows to producehetero-oligomeric proteins having more than two different subunits, e.g.three or four different protein subunits. In this case, said plant,plant tissue or plant cells may be provided with a first, a second, anda third viral vector (and optionally with a further viral vector), eachencoding one of said different protein subunits. If three or fourdifferent protein subunits have to be expressed in step (i), therespective three or four viral vectors are preferably all non-competingviral vectors with each other. In the case of three viral vectors, thefirst viral vector may be derived from a tobamovirus, the second viralvector may be derived from a potyvirus, and the third viral vector maybe derived from a potexvirus.

Two viral vectors are non-competing if they can express efficiently theprotein subunits they encode in the same plant cell (co-expression).Co-expression requires that the at least two different viral vectors donot outcompete each other during replication before having expressedsubstantial amounts of the heterologous sequences they encode. Thehigher the sequence differences on the RNA level of said at least twoviral vectors, the more they are non-competing in the same plant cells.Said first viral vector and said second viral vector are non-competingviral vectors, since said first viral vector and said second viralvector are different viral vectors.

For being non-competing, said first and said second viral vectors (saidtwo non-competing viral vectors) differ, in one general embodiment, atleast in a sequence portion other than said heterologous sequencescoding for said protein subunits. Preferably, such a difference in asequence portion other than said heterologous sequences is a sequenceportion derived from a plant RNA virus such as a sequence portioninvolved in a viral function such as viral replication in plant cells(e.g. a sequence coding for a replicase), translation of a viral protein(e.g. a subgenomic promoter) or viral cell-to-cell or long distancemovement.

For being non-competing, said first and said second viral vectors (saidtwo non-competing viral vectors) are, in another general embodiment,derived from different plant viruses. In one example of this embodiment,said at least two non-competing viral vectors are not derived fromviruses of the same virus strain. In another example, said at least twonon-competing viral vectors are not derived from viruses of the samevirus species. In a further example, said at least two non-competingviral vectors are not derived from viruses of the same virus genus.Thus, said first viral vector and said second viral vector arepreferably derived from viruses of different strains, more preferablyfrom viruses of different species, most preferably from viruses ofdifferent genera.

Said first viral vector may be derived from a virus belonging to thegenus Potexvirus and said second viral vector may be derived from avirus belonging to the genus Potyvirus. Specifically, said first viralvector may be derived from Potato Virus X and said second viral vectormay be derived from Potato Virus Y.

In case of a potyviral vector, said protein subunit can be expressed asa fusion with a viral polyprotein, whereby a protein subunit of theinvention can be separated from said polyprotein by a potyviral proteaserecognition site.

In another embodiment, said first viral vector may be derived from avirus belonging to the genus Potexvirus and said second viral vector maybe derived from a virus belonging to the genus Tobamovirus.Specifically, said first viral vector may be derived from Potato Virus Xand said second viral vector may be derived from Tobacco Mosaic Virus.

“Being derived from a virus” means that the viral vector containsgenetic elements or sequence portions from the virus it is derived from.In one embodiment, said viral vector(s) contain(s) a replicase ORF (openreading frame) taken from an RNA virus. In another embodiment, a viralvector contains a movement protein ORF from an RNA virus and,optionally, also a replicase ORF. Viral genetic elements taken from anRNA virus may, if desired, be mutated e.g. for introducing therestriction sites required for cloning of the viral vector.

An embodiment wherein said first viral vector and said second viralvector are both based on tobacco mosaic viral vector TMV 30 B isexcluded from step (i) of the process of the invention, since in thiscase said first viral vector and said second viral vector are notdifferent viral vectors but are the same viral vectors (cf. Verch etal., J. Immunological Methods 220 (1998) 69-75).

Said first and said second viral vectors (said non-competing viralvectors) preferably have a sequence homology on RNA level of at most90%, more preferably of at most 80%, even more preferably of at most70%, and most preferably of at most 60%. More specifically, any sequencesegment of said first viral vector of 100 bases preferably has asequence homology to any sequence segment of 100 bases of said secondviral vector of at most 90%, preferably at most 80%, more preferably atmost 70%, and most preferably of at most 60%.

In one embodiment, the replicase ORF (or ORFs if the replicase isencoded by more than one ORF) of said first viral vector and thereplicase ORF (or ORFs if the replicase is encoded by more than one ORF)of said second viral vector have a homology of at most 90%, morepreferably of at most 80%, even more preferably of at most 70%, and mostpreferably of at most 60%. In another embodiment, the replicase ORF ofsaid first viral vector and the replicase ORF of said second viralvector have an identity of at most 85%, more preferably of at most 75%,even more preferably of at most 65%, and most preferably of at most 55%.

In step (i), said first viral vector preferably contains a firstheterologous sequence encoding said first protein subunit expression ofwhich may be under the control of a first sub-genomic promoter. Saidsecond viral vector contains a second heterologous sequence encodingsaid second protein subunit expression of which may be under the controlof a second subgenomic promoter. One or both of said subgenomicpromoters may be replaced by an IRES element. Similar as described abovefor step (ii), if both said first and said second protein subunits areexpressed under the control of a subgenomic promoter, these subgenomicpromoters preferably differ in sequence for avoiding homology betweensaid first and said second (and any further viral vector) viral vector,which could lead to undesired recombination events in plant cells. Suchdifferent subgenomic promoters may be taken from different strains orspecies of plant virus, e.g. one subgenomic promoter may the coatprotein (CP) subgenomic promoter of tobacco mosaic virus (TMV) U1 andthe other subgenomic promoter may be the CP subgenomic promoter of TMVU5 or from crucifer-infecting tobamovirus (cr-TMV).

Since ORFs of plant viruses that are required for cell-to-cell or longdistance movement may be omitted upon constructing the viral vectors ofthe invention, the relatedness of said non-competing viral vectors maybe determined by comparing the replicase ORFs of said viral vectors.

Said first and said second heterologous sequences encoding said firstand said second protein subunit, respectively, may be added as anadditional sequence to said viral vectors. Said heterologous sequencesare preferably added such that high level expression is achieved. Forthis purpose, said heterologous sequence is added at the 3′ end of thevirus, since the 3′ ORF is frequently the ORF that is expressed at thehighest level in many viruses. Preferably, however, said heterologoussequences replace a sequence native to said virus, e.g. the natural 3′ORF of said virus which is the CP ORF in many viruses liketobamoviruses. Thus, said first and/or said second viral vectorpreferably lacks an ORF for systemic movement of said viral vector. Saidviral vectors may further lack an ORF for cell-to-cell movement like theMP ORF in tobamoviruses.

In this invention, step (i) and step (ii) may be combined, notably forexpressing three or more different subunits of a hetero-oligomericprotein. If a hetero-oligomeric protein having four different proteinsubunits is to be produced, two protein subunits may be expressedaccording to step (i) and two further protein subunits may be producedin the cells of a plant or plant tissue according to step (ii).Preferably, however, two protein subunits may be expressed from a firstviral vector and two protein subunits may be expressed from a secondviral vector that is non-competing to the first viral vector. If ahetero-oligomeric protein having three different protein subunits is tobe produced, said three protein subunits may be expressed according tostep (i) by expressing two proteins from a first viral vector similarlyas described for step (ii) and expressing a third protein subunit from anon-competing viral vector.

The viral vectors of the invention are typically engineered on DNAlevel. If said viral vectors are provided to cells of a plant or tocells of plant tissue as RNA viral vectors, said DNA may be transcribedin vitro to said RNA viral vectors e.g. using a bacteriophage polymeraselike T7 polymerase together with a suitable promoter. Two differentviral vectors are preferably applied to said plant as a mixture forensuring that cells of said plant are provided with both viral vectors.Preferably, however, the viral vectors of the invention are provided tocells of a plant or to cells of plant tissue by transforming said plantor said plant tissue with DNA precursors of said RNA viral vectors. SaidDNA precursors have a transcriptional promoter active in cells of saidplant for forming said viral vectors by transcription of said DNAprecursors. Most preferably, said DNA precursors are T-DNA inAgrobacterial Ti plasmids. Two or more viral vectors may then beprovided to said plant or said plant tissue by treating said plant witha mixture (e.g. a suspension) of two or more Agrobacterium strains,whereby each strain contains a T-DNA encoding a particular viral vector.Treating substantial parts of a plant with such an Agrobacteriumsuspension may replace the systemic movement function and/or thecell-to-cell movement function of natural plant viruses.

Transient transfection of said plant, plant tissue or plant cells withDNA precursors of said viral vectors by way of Agrobacterium is mostpreferred in the present invention. However, said DNA precursors of saidviral vectors may be stably incorporated into plant chromosomal DNA.Release of said viral vector(s) from chromosomal DNA may be controlledby inducible promoters.

If said viral vectors are provided to said plant by way of DNAprecursors, it is preferred that measures are taken for improving theefficiency of transfer of said viral vectors from the cell nuclei wherethey are transcribed to the cytoplasm where said viral vectorsreplicate. This may be achieved by including introns in said DNAprecursors, notably in the replicase ORFs of the viral vectors asdescribed in detail in International patent application PCT/EP05/000492,published as WO2005/71090, that is incorporated herein by reference.

The process of the invention may be applied to any plant for which plantviral expression systems exist or will be worked out in the future. Saidplant may be a monocot or a dicot. Among dicots, Solanaceae,Brassicaceae, Chenopodiaceae, and Legume are preferred. AmongSolanaceae, the genus Nicotiana like N. tabacum or N. benthamiana ispreferred. Other preferred plants are Medicago sativa and Beta specieslike Beta vulgaris.

The process of the invention is used for producing hetero-oligomericproteins in plant systems. Preferred hetero-oligomeric proteins areimmunoglobulins like immunoglobulins of the following classes:immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin D,and immunoglobulin E. These immunoglobulins may comprise at least aportion of an antigen binding domain. As the case requires, theseimmunoglobulins produced according to the invention may be modifiedrelative to native animal immunoglobulins, provided they comprise atleast two different protein subunits. The immunoglobulin may comprises aprotection protein in association with an immunoglobulin heavy chain,wherein the protection protein comprises a portion of apolyimmunoglobulin receptor. Another preferred hetero-oligomeric proteinis insulin.

The hetero-oligomeric protein of the invention may be modified in manydifferent ways relative to the native protein as the case requires. Inthe hetero-oligomeric protein, a native leader sequence forming asecretion signal of one or more protein subunits of the nativehetero-oligomeric protein may be replaced by plant-specific signalpeptides. Said plant-specific signal peptides may be derived fromtobacco calreticulin and/or rice alpha-amylase. At least one or at leasttwo or more subunits of said hetero-oligomeric protein may contain anendoplasmatic reticulum retention signal KDEL for improving assembly ofsaid hetero-oligomeric protein from said subunits in plant cells.Further, said heterologous sequences encoding said protein subunits maybe mutated in order to partially or completely remove glycosylationsites from said hetero-oligomeric protein. Moreover, the glycosylationpattern of the hetero-oligomeric protein to be expressed may be changede.g. by engineering a component of the plant glycosylation machinerylike one or more glycosyl transferases.

The hetero-oligomeric protein of the invention may be isolated from theplant, plant tissue or plant cells after expression according togenerally known procedures. Said hetero-oligomeric protein may then bepurified to substantial homogeneity, which state may be defined suchthat bands due to said hetero-oligomeric protein on a coomassie-stanedSDS-PAGE account for at least 70%, preferably at least 80%, and mostpreferably at least 90% of the staining of a lane as determined by aconventional gel reader.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an expression distribution pattern of GFP and DsRed fromtwo different TMV-based vectors in infiltrated N. benthamiana leaves.Left picture: the light spot at the top left side is GFP fluorescence ofan area infiltrated with pICH17272. The weakly light spot below is redfluorescence of an area infiltrated with vector pICH18505. The lightspot at the bottom right side of the left picture is an area infiltratedwith pICH17272+pICH18505. The pictures on the right hand side showprotoplasts isolated from a leaf area co-infiltrated with two differentvectors (top: protoplasts under GFP fluorescence detection conditions;bottom panel: the same protoplasts under DsRed fluorescence detectionconditions.

FIG. 1B is a schematic representation of T-DNA regions of pICH17272 andpICH18505. P—transcription promoter; T—transcription termination region;RdRP viral RNA-dependent RNA polymerase; MP—viral movement protein;3′NTR—viral 3′ non-translated region; Cr-sgp—CP subgenomic promoterregion of crTMV strain; GOI—gene of interest.

FIG. 1C are schematic representations of restriction maps of the T-DNAregions of pICH17272 and pICH18505.

FIG. 2 depicts a schematic representation of a T-DNA region of a viralvector designed for co-expression of two different transgenes (GOI-1 andGOI-2) from different subgenomic promoters. P—transcription promoter;T—transcription termination region; RdRP viral RNA-dependent RNApolymerase; MP—viral movement protein; 3′NTR—viral 3′ non-translatedregion; Cr-sgp—CP subgenomic promoter region of crTMV strain; 3PK—triplepseudo knot region; U5sgp—CP subgenomic promoter of TMV-U5 strain.GOI—gene of interest.

FIG. 3A depicts schematic representations of T-DNA regions of constructspICH17388, pICH17123, pICH15933, pICH7410, pICH10580, pICH10881 andpICH10745. RS—recombination site recognised by PhiC31 integrase. Greyvertical bars indicate introns.

FIG. 3B depicts restriction maps of the T-DNA regions of pICH17388,pICH17123, pICH15933.

FIG. 3C depicts restriction maps of the T-DNA regions of pICH7410,pICH10580, pICH10881 and pICH10745.

FIG. 4A shows in fluorescence microscope micrographs of an N.benthamiana leaf region 6 days after agrobacterial delivery of DNAprecursors pICH17388 and pICH15933 together with a recombinase source.

FIG. 4B depicts schematic representations of constructs pICH17388 andpICH15933. RS—recombination site recognised by PhiC31 integrase.

FIG. 5 depicts schematic representations of T-DNA regions of differentvector systems for the expression of heavy and light chains of anantibody. RS—recombination site recognised by PhiC31 integrase.

FIG. 6 shows a Western blot of the expression of an IgG in Nicotianabenthamiana leaves using viral provector system.

Electrophoretic separation of TSP was carried out on a 12% gel undernon-reducing conditions. Detection of expressed protein was performedwith anti-human IgG fraction from rabbit conjugated with HRP (Sigma)diluted 6×10³.

Lane: 1—uninfected leaf tissue; lane 2—IgG heavy chain expressed incytosol (pICH17388); lane 3—IgG heavy and light chains, co-expressionwith 35S promoter constructs (pIC0123+pICH19846); lane 4—the same aslane 3 with P19 (pICH6692); lane 5—GFP expressed with 35S promoterconstructs and P19 (pICH5290+pICH6692); lane 6—IgG heavy and lightchains co-expression with bicistronic construct pICH19860; lane 7—IgGheavy and light chains co-expression with bicistronic constructpICH19860 (MP deficient 5′ pro-vector pICH17123, MP in trans pICH10745);lane 8—GFP and dsRED co-expressed in cytosol, MP provided in trans(pICH17123+pICH19919+pICH10745).

FIG. 7 depicts a schematic representations of T-DNA regions encodingnon-competing viral vectors designed for co-expression of differentprotein subunits of interest in the same plant cell. P—transcriptionpromoter; T—transcription termination region; TMV RdRP viralRNA-dependent RNA polymerase of Tobacco Mosaic Virus; PVX RdRP viralRNA-dependent RNA polymerase of Potato Virus X; MP—viral movementprotein; 3′NTR—viral 3′ non-translated region; Cr-sgp—CP subgenomicpromoter region of crTMV strain; GOI—gene of interest coding for aprotein subunit of interest.

FIG. 8 depicts a schematic representation of the T-DNA region of binaryvector pIC0130.

FIG. 9 shows co-expression of GFP and dsRED in plant cells using TMV(pICH17388+pICH10580) and PVX-based (pIC0130) vectors: visualisation ofGFP and dsRED in protoplast isolated from agroinoculated N. benthamianaleaves.

FIG. 10A depicts schematic representation of the T-DNA regions of binaryvectors pICH17620, pICH20431, pICH21240, pICH20421, and pICH21370.

FIG. 10B is a schematic representation of the T-DNA regions of binaryvectors pICH11599, pICH21910 and pICH21920.

FIG. 10C is a schematic representation of the T-DNA regions of binaryvectors pICH21282, pICH10990, pICH22250 and pICH22240. Lv andLc—variable and conservative regions of light chain; Hv and Hc—variableand conservative regions of heavy chain.

FIG. 10D is a schematic representation of cloning scheme for PVX-derivedprovector pICH21380.

FIG. 11A shows the results of an ELISA test for the determination ofco-expression levels of IgG light and heavy chains using PVX and crTMVvectors. The numbering of the wells of the tissue culture plate on theleft corresponds to the numbering of the bars in the histogram. Thehistogram displays the OD of the wells at 405 nm.

1,2—uninfected plant tissue;

3,4,5—calreticulin SP-Heavy Chain in PVX (pICH21240-1, 5 and 14 clones,respectively);

6,7,8—calreticulin SP-Light Chain (LC) of IgG in PVX (deletion inN-terminus, pICH21370-18, 19, 31, respectively);

9, 10, 11—calreticulin SP-Light Chain of IgG in PVX (pICH21370-40, 44,45, respectively); 12-blanc control (no plant protein extract applied);

13, 14, 15—calreticulin SP-Heavy Chain (HC) of IgG in PVX (pICH21240-1,5 and 14 clones, respectively) co-expressed with calreticulin SP-LightChain of IgG in crTMV (pICH 17620+pICH10881+pICH20431);

16, 17, 18—calreticulin-LC of IgG in PVX (deletion in N-terminus,pICH21370-18, 19, 31, respectively) co-expressed with calreticulin-HC incrTMV (pICH 17620+pICH10881+pICH20421);

19, 20, 21—calreticulin SP-LC of IgG in PVX (pICH21370-40, 44, 45,respectively) co-expressed with calreticulin SP-Heavy Chain incrTMV-based vector (pICH 17620+pICH10881+pICH20421);

22—GFP expressed with PVX (pIC0130);

23—GFP expressed with PVX (pICH20799);

24—calreticulin SP-Light Chain of IgG expressed from crTMV alone (pICH17620+pICH10881+pICH20431);

25—calreticulin SP-Heavy Chain of IgG expressed from crTMV-based vector(pICH 17620+pICH10881+pICH20421);

26—Light and Heavy Chains of IgG co-expressed under control of strong35S promoter at presence of PTGS suppressor P19;

27—IgG Light and Heavy Chains co-expression from crTMV-based vector(pICH17388+pICH10881=pICH20241);

28—Light and Heavy Chains co-expression from crTMV-based vector(pICH17388+pICH10881=pICH20241), MP (pICH10745) provided in trans;

*—OD405 value well above of measurable values.

FIG. 11B-11D shows electrophoretic and Western blot analysis ofanti-cancer antibodies expressed in N. benthamiana leaves.

FIG. 11B Accumulation of heavy and light chains of an anti-cancerantibody in Nicotiana benthamiana leaves co-infected with TMV and PVXpro-vectors. Light chain is expressed with PVX and heavy chain with TMV.Proteins are separated in 12% polyacrylamide gel under reducingconditions. The upper panel shows coomassie staining; the middle panelshows a Western blot with HRP-conjugated goat anti-human IgG (gammachain-specific) antibodies (Sigma); the lower panel shows a Western blotwith HRP-conjugated rabbit anti-human IgG (lambda chain-specific)antibodies (Sigma). Uninf, uninfected tissue; 2-11, days postinoculation; S, anti-cancer mab (monoclonal antibody) standard; LC,light chain expressed alone with TMV pro-vectors (6 dpi); HC, heavychain expressed alone with TMV pro-vectors (6 dpi).

FIG. 11C Purification of anti-cancer mab using Protein A Magnetic beads(NEB). (A) Plant-derived (lane 1) and standard (lane 2) mabs migratingin 12% polyacrylamide gel under the non-reducing conditions on Coomassiestained gel.

FIG. 11D Accumulation of assembled anti-cancer mab in Nicotianabenthamiana leaves co-infected with TMV pro-vector expressing the HC andPVX pro-vector expressing the LC. Proteins are separated in 10%polyacrylamide gel under the non-reducing conditions. The Western blotwas probed with goat anti-human IgG (gamma chain-specific) antibodies.Uninf, uninfected tissue; 2-11, days post inoculation; S, anti-cancermab standard; LC, light chain expressed alone with TMV pro-vectors (6dpi); HC, heavy chain expressed alone with TMV pro-vectors (6 dpi).H₂L₂: IgG heterotetramer containing two heavy and two light chains,H2L—heterotrimer containing two heavy and one light chains, H2—heavychain homodimer, L2—light chain homodimer.

FIG. 12 shows a schematic representation of T-DNA regions of binaryvectors pICH17620, pICH-FSHA and pICH-FSHB.

FIG. 13 depicts a schematic representation of T-DNA regions of binaryvectors pICH17388, pICH-MLCJ and pICH-MHC.

DETAILED DESCRIPTION OF THE INVENTION

The viral vectors described in present invention are either viralvectors wherein a single vector encodes all protein subunits necessaryfor forming said hetero-oligomeric protein, or at least two differentnon-competing viral vectors, whereby each of said at least two viralvectors encodes for different protein subunit necessary for forming saidhetero-oligomeric protein. Each of said non-competing viral vectors canexpress more than one heterologous nucleic acid sequence encoding morethan one subunit of recombinant hetero-oligomeric protein. Said RNAviral vectors can be transiently delivered into plant cell or can bestably incorporated into plant chromosomal DNA as DNA precursor(s).

The present invention provides a process for high-yield production ofhetero-oligomeric proteins in plant cells. This process overcomes thelimitations of existing viral vector-based expression systems, such assize limitation for heterologous sequences to be expressed, highinstability of said vectors and inability to co-express differentheterologous nucleic acid sequences in the same plant cell. Further,said process offers better biosafety characteristics, as the removal ofviral coat protein from the system prevents formation of infectiousviral particles and reversion to wild type viruses. By practicing theinvention, the design of high-yield expression system forhetero-oligomeric protein of interest is possible for practically anyplant RNA virus-derived replicon, said replicon is suitable for theexpression of a heterologous sequence of interest, through modificationof said replicon to be capable expressing at least two heterologoussequences of interest encoding for different subunits ofhetero-oligomeric protein of interest. Alternatively, anothernon-competing viral vector can be found that is able to co-replicatewith said viral vector in the same plant cell.

Plus-sense single-stranded RNA viruses (also referred to herein as “RNAviruses” for brevity) belonging to different taxonomic groups aresuitable for constructing the plus-sense single-stranded RNA viralvectors (also referred to herein as “viral vectors” for brevity) of thisinvention. Herein, a viral vector is an RNA vector capable ofreplicating in plant cells, i.e. forming further RNA vector molecules byRNA-dependent RNA polymerization using the RNA viral vector as atemplate. Preferably, the viral vectors of the invention contain atleast one iral sequence element having an RNA viral function e.g. areplicase, a subgenomic promoter, an origin of viral particle assembly,a coat protein ORF, or a movement protein ORF. Further, the viral vectormay have an RNA viral IRES element.

A viral vector can e.g. be constructed from the virus it is derived fromby introducing restriction sites into the virus, said restriction sitesrepresenting cloning sites suitable for introducing the heterologoussequence of the invention. It will be understood by the skilled personthat nucleic acid engineering on RNA viruses or vectors is generallydone on the DNA level using DNA copies of said RNA viruses or vectors,respectively. Thus, it is more accurate to say that a DNA copy of an RNAviral vector can be constructed from the DNA copy of the RNA virus it isderived from by introducing restriction sites into the DNA copy of theRNA virus, said restriction sites representing cloning sites suitablefor introducing the DNA copy of the heterologous sequence of theinvention on the DNA level. These matters are obvious for a skilledperson and will in general not be stressed herein.

If a DNA copy of an RNA virus naturally has (a) restriction site(s)suitable for cloning, the virus itself may be a viral vector. Herein,the term “viral vector” refers to the case where said heterologoussequence is not present in said vector or to the case where no sequencecoding for a protein subunit of the invention is present in the vector.Cases wherein said heterologous sequence or a sequence coding for aprotein subunit of the invention is inserted into said viral vectors areidentified by explicitly specifying the presence of such insert.

Herein, two viral vectors are referred to as being different, if theirsequences are different before said heterologous sequences or a sequencecoding for a protein subunit of the invention have been introduced. Twoviral vectors are referred to as being the same, if they have the samesequences before said heterologous sequences or a sequence coding for aprotein subunit of the invention have been introduced. Thus, saidheterologous sequences or sequences coding for the protein subunits ofthe invention are not taken into account when determining whether twoviral vectors are different or the same.

Herein, the terms “replicon” or “viral replicon” have the same meaningas “viral vector”.

A list of RNA viruses that can be used for engineering the viral vectorsof the invention is presented below. Taxa names in quotes (and not initalic script) indicate that this taxon does not have an ICTVinternational approved name. Species (vernacular) names are given inregular script. Viruses with no formal assignment to genus or family areindicated):

RNA Viruses:

ssRNA Viruses:

Family: Bromoviridae,

Genus: Alfamovirus, Type species: alfalfa mosaic virus,Genus: Ilarvirus, Type species: tobacco streak virus,Genus: Bromovirus, Type species: brome mosaic virus,Genus: Cucumovirus, Type species: cucumber mosaic virus;

Family: Closteroviridae,

Genus: Closterovirus, Type species: beet yellows virus,Genus: Crinivirus, Type species: Lettuce infectious yellows virus,

Family: Comoviridae,

Genus: Comovirus, Type species: cowpea mosaic virus,Genus: Fabavirus, Type species: broad bean wilt virus 1,Genus: Nepovirus, Type species: tobacco ringspot virus; Family:Potyviridae,Genus: Potyvirus, Type species: potato virus Y, plum pox virus; tobaccoetch virus; cloveryellow vein virus; tobacco vein mottling virus;Genus: Rymovirus, Type species: ryegrass mosaic virus,Genus: Bymovirus, Type species: barley yellow mosaic virus;

Family: Sequiviridae,

Genus: Sequivirus, Type species: parsnip yellow fleck virus, Genus:Waikavirus, Typespecies: rice tungro spherical virus;

Family: Tombusviridae,

Genus: Carmovirus, Type species: carnation mottle virus, Genus:Dianthovirus, Typespecies: carnation ringspot virus, Genus: Machlomovirus, Type species:maize chloroticmottle virus, Genus: Necrovirus, Type species: tobacco necrosis virus,Genus:Tombusvirus, Type species: tomato bushy stunt virus,Unassigned Genera of ssRNA viruses,Genus: Capillovirus, Type species: apple stem grooving virus; Genus:Carlavirus, Typespecies: carnation latent virus;Genus: Enamovirus, Type species: pea enation mosaic virus,Genus: Furovirus, Type species: soil-borne wheat mosaic virus,Genus: Hordeivirus, Type species: barley stripe mosaic virus,Genus: ldaeovirus, Type species: raspberry bushy dwarf virus;Genus: Luteovirus, Type species: barley yellow dwarf virus;Genus: Marafivirus, Type species: maize rayado fino virus;Genus: Potexvirus, Type species: potato virus X;Genus: Sobemovirus, Type species: Southern bean mosaic virus,Genus: Tenuivirus, Type species: rice stripe virus,Genus: Tobamovirus, Type species: tobacco mosaic virus,Genus: Tobravirus, Type species: tobacco rattle virus,Genus: Trichovirus, Type species: apple chlorotic leaf spot virus;Genus: Tymovirus, Type species: turnip yellow mosaic virus;Genus: Umbra virus, Type species: carrot mottle virus;Negative ssRNA Viruses: Order: Mononegavirales, Family: Rhabdoviridae,Genus: Cytorhabdovirus, Type Species: lettuce necrotic yellows virus,Genus: Nucleorhabdovirus, Type species: potato yellow dwarf virus.

RNA viral vectors are able to provide an extremely high copy number ofheterologous RNA providing for expression of the gene of interest inplant cell. However, it is known that such vectors become extremelyunstable, if the size of heterologous nucleic acid sequence is increasedbeyond certain limits, usually beyond 1 kb. Due to such limitations theapplication of such vector systems has so far been restricted to theexpression of relatively simple small to medium sized proteins. Attemptsto express either large or complex hetero-oligomeric proteins have notled to a successful outcome. We have surprisingly found that viralvectors can be successfully adopted for the high-yield expression ofcomplex hetero-oligomeric proteins, which has not been possible before.Early attempts to express full length monoclonal antibody (Verch et al.,1998, J. Immunol. Meth., 220, 69-75) did not provide satisfactoryresults due to the incompatibility of the viral vectors used forco-expression of proteins of interest in the same cell. In Example 1 wedemonstrate on single cell level that efficient co-expression of twodifferent genes (GFP and DsRed) from viral replicons derived from thesame plant RNA virus (TMV) is not possible. In FIG. 1 (A-right panel) wecould not detect protoplasts that show expression of both reportergenes—DsRed and GFP. A weak expression pattern of DsRed in someprotoplasts at right bottom panel coincides with strong GFP expression(right top panel) and is a false-positive result due to leakage of thefilter used for DsRed detection. This leakage leads to an apparent weakDsRed fluorescence of protoplasts containing high concentrations of GFP.Thus, RNA replicons which are identical or share extensive regions ofhomology cannot co-exist in one plant cell even when they carrydifferent heterologous nucleic acid sequences encoding for differentrecombinant proteins. The reason for this phenomenon is presently notknown. A possible explanation is that the exponential increase in copynumber of one viral replicons results in quick outcompetition of theother viral replicon. So a replicon which is second to start replicationin a selected cell cannot catch up with the first one, whereby theevents determining the “first” replicon are predominantly of statisticalcharacter.

In our studies to address this problem, we have engineered a viralreplicon such that two different heterologous nucleic acid sequencesencoding different proteins under control of two different subgenomicpromoters are present in said replicon. The general scheme of a T-DNAregion encoding such an RNA replicon is shown in FIG. 2. As a matter ofconvenience for design and optimisation of such a vector, we have usedthe pro-vector approach described in our earlier patent application(WO02088369; see also Marillonnet et al., 2004, Proc. Natl. Acad. Sci.USA, 101, 6852-6857). This approach allows to assemble in planta thefinal vector from pre-made modules via site-specific recombination, thussignificantly speeding up vector design and vector optimisation. Thedesign of the constructs is described in Example 2 and schematicrepresentations are shown in FIG. 3.

We have surprisingly found that despite of the larger size of the insertin the final replicon and the complex structure of the vector due to thepresence of two strong subgenomic promoters, the obtained RNA repliconshowed high stability in planta and the ability to provide forco-expression of two different recombinant proteins (GFP and DsRed) inthe same plant cell. As is shown in FIG. 4, the co-expression frequencycan reach up to almost 100% of all cells in infected areas. Replacementof reporter genes (GFP and DsRed) e.g. with the light and heavy chainsof an IgG in such a construct (FIG. 5) with further expression ininfiltrated N. benthamiana leaves produced a surprising results. As isdescribed in Example 3, a Western blot analysis revealed an impressivelyhigh concentration of assembled monoclonal antibodies (lanes 6 and 7;FIG. 6). The yield of assembled monoclonal antibody provided by oursystem is incomparably higher than that produced by current state of artsystem (FIG. 6, lane 4). To our knowledge, this is the first evidence ofthe expression of a complex hetero-oligomeric protein like an antibodyusing a plant viral vector-based expression system. All earlierpublications were restricted to the expression of simple artificialderivatives of monoclonal antibodies, e.g. to the expression of singlechain antibodies (scFv) using TMV-viral vectors (McCormick et al., 1999,Proc Natl Acad Sci USA, 96, 703-708; McCormick et al., 2003, J. Immunol.Methods, 278, 95-104) and PVX-based (Smolenska et al., 1998, FEBS Lett.,441, 379-382; Franconi et al., 1999, Immunotechnology, 4, 189-201; Hendyet al., 1999, J. Immunol. Methods, 231, 137-146; Roggero et al., 2001,Protein Expr. Purif., 22, 70-74).

In this invention, we preferably do not use systemic viral vectors.Instead of systemic viral vectors, we preferably replace the ability ofthe viral vector to move systemically by agrobacterium-mediated deliveryof viral vector precursors into the plant system. This allows us toreplace the viral coat protein with a heterologous sequence to beexpressed. This approach contributes to the possibility of increasingthe capacity of the viral vector for heterologous sequences. At the sametime, this approach eliminates the probability of the viral vector to beconverted to a wild type virus due to spontaneous deletion ofheterologous sequences, which would compromise the productivity of thesystem.

Although our viral replicon-based system produced significantly betteryield of monoclonal antibody than prior art systems, we examined thepossibility of further increase the yield by decreasing the size of theviral replicon. Considering that there are limited possibilities todecrease the size of a viral replicon expressing two chains of asecretiory antibody, we have attempted the expression of heavy and lightantibody chains from two different viral replicons. Considering thatreplicons based on the same virus are not able co-express two differentheterologous sequences in the same plant cell (see above, example 1), wetested the possibility of co-expressing two different genes in the sameplant cell by separately cloning DsRed and GFP genes into viral vectorsderived from the non-homologous plant viruses tobacco mosaic virus (TMV)and Potato Virus X (PVX) (see Example 4). Schematic representation ofT-DNA regions containing cDNAs of said viral vectors is shown in FIGS. 7and 8 (PVX only). In Example 4, as a matter of convenience, we used aTMV-based viral vector assembled in planta from vector modules viasite-specific recombination. Surprisingly, we have found thatprotoplasts isolated from a co-infiltrated plant leaf region showedpractically 100% co-expression frequency of DsRed and GFP reporter genes(FIG. 9).

Recently, a study on the spatial separation of differently labelledviruses was presented by Dietrich and Maiss (2003, J. Gen. Virology, 84,2871-2876). In this study, fluorescent proteins were expressed asreporters. Production of hetero-oligomeric proteins was not mentioned.Further, co-expression at the level of isolated protoplasts was notinvestigated. Since reporter gene products can diffuse to neighbouringcells via diffusion through plasmodesmata, no information was availablewhether the selected pairs of viruses expressed the reporter genes inthe same or in neighbouring cells. The earlier publications relate tosynergism between wild type viruses upon infection of plants, but do notrelate to expression of hetero-oligomeric proteins in plant cells(Rochow & Ross, 1955, Virology, 1, 10-27; Goodman & Ross, 1974,Virology, 58, 16-24; Goodman & Ross, 1974, Virology, 59, 314-318;Goodman & Ross, 1974, Virology, 58, 263-271). Later development of theseearly studies on synergistic interactions of viruses in co-infectedplants (Vance et al., 1995, Virology, 206, 583-590; Pruss et al., 1997,Plant Cell, 9, 859-868) led to the discovery of suppressors ofpost-transcriptional gene silencing (PTGS). The further development ofrecombinant protein expression systems was directed to study of suchPTGS suppressors for enhanced production of recombinant proteins. Thereis no hint in the prior art to protein expression using viral expressionsystems based on synergistic viruses.

This invention demonstrates that non-competing viral vectors representan efficient tool for the production of hetero-oligomeric proteins inplant cells or plants. Two or more viral vectors are non-competing witheach other, if said viral vectors are capable of replicating in the sameplant cell and do not dilute each other during said replication andtransfection of other cells. Absence of dilution means that at least10%, preferably 50%, more preferably 90%, and most preferably 100% oftransfected plant cells are co-transfected, e.g. contain said two ormore different viral vectors. Preferably, said viral vectors are capableof replicating and expressing heterologous nucleic acid sequences. Morepreferably, said heterologous nucleic acid sequences encode fordifferent proteins. Even more preferably, said different proteins aresubunits of hetero-oligomeric protein. In order to determine whetherviral vectors are competing or non-competing, the frequency ofco-transfection of plant cells can be measured. This can be done by thefollowing protocol: two viral vectors are labelled with two differentreporter genes, e.g. DsRed and GFP. Said differently labelled vectorsare co-delivered (e.g. via agro-infiltration) into plant leaf tissue and3-6 days later, the protoplasts isolated from the infected region can becounted in order to determine the proportion of protoplastsco-expressing both reporter genes compared to the number of protoplastsexpression only one reporter gene. Experiments demonstrating suchmeasurements are described in Examples 2 and 4 (see also the FIGS. 1Aand 9). Usually, non-competing viral vectors can derive from virusesthat are synergistic, e.g. can successfully co-transfect the same planthost. Examples of such pairs of synergistic RNA viruses include:

Potato Virus X (PVX)/Tobacco Mosaic Virus (TMV); PVX/Tobacco VeinMottling Virus (TVMV); PVX/Tobacco Etch Virus (TEV);

PVX/Clover Yellow vein virus (CIYVV);

PVX/Plum Pox Virus (PPV); PVX/Potato Virus Y (PVY).

The mechanistic reason for competitiveness or non-competitivness ofviral vectors is not known. One possible explanations is that viralreplicons derived from synergistic viral vectors form viral replicationcomplexes (VRCs) (Kawakami et a!, 2004, Proc. Nat!. Acad. Sci. USA, 101,6291-6296) at different sub-cellular compartments, thus they are notcompeting with each other for the space necessary for forming VRCs. Asit was established experimentally, non-competing viral vectors may bederived from different species of viruses that are significantlydifferent at the level of their nucleic acid sequences. Saidnon-competing viral vectors can be derived from synergistic plantviruses that can successfully co-infect the same plant host. Synergisticviruses usually belong to different genuses. For example, PVX belongs tothe genus Potexvirus, while viruses synergistic thereto like TVMV, TEV,PPV, and CIEVV belong to the genus Potyvirus. Other viruses synergistic(non-competing) to PVX viruses, like TMV, TVCV, and crTMV belong to thegenus Tobamovirus. Obviously, the genome homology betweenrepresentatives of different genuses is rather low, usually below 50%identity on the RNA level.

Cloning of heavy and light chains of a monoclonal antibody intodifferent viral vectors (e.g. PVX and TMV-based) following furtherco-expression of said chains in co-infected plant tissue is described inExample 5. Schematic representations of the vectors is shown in FIG. 10.Comparative ELISA measurement of yield of monoclonal antibodies producedwith help of this and other expression systems showed the bestperformance of said system based on two non-competing viral vectors(FIG. 11). Efficient expression of recombinant hetero-oligomericproteins in plant cell with the help of viral vector(s) is not known inthe prior art.

Based on our data, virus-derived sequences of the viral vectors shallnot exhibit homology significant enough to consider said viruses relatedto each other. An example of such viruses is the virus pair TMV and PVXand viral vectors based thereon. These viral vectors exhibit no suchhomology. Another requirement for selecting acceptable virus pairs canbe the synergism of the original wild type viruses during co-infectionof a plant host.

The experiments discussed above were done with transient expressionsystems based on Agrobacterium-mediated DNA precursor delivery intoplant cells. However, an alternative application of this invention isfor transgenic plants with a DNA precursor of said RNA replicon(s)stably incorporated into a plant nuclear chromosome. This allows toovercome many limitations of plant viral vector-based systems, such asthe restrictions to the maximal size of heterologous sequences viralvectors can tolerate. As the DNA precursor will be present in each cellof the transgenic plant, there is no absolute requirement for systemicmovement or for cell to cell movement of the RNA replicon (repliconspreading). This can be compensated by the high efficiency of formationand transport of the RNA replicons of the invention into the cytoplasm.However, the ability of the vector for cell-to-cell movement can be ofan additional value, as RNA replicon formation does not always occur inall cells.

Different methods may be used for providing a plant, plant tissue orplant cells with heterologous DNA. Said vectors may be transformed intoplant cells by a Ti-plasmid vector carried by Agrobacterium (U.S. Pat.No. 5,591,616; U.S. Pat. No. 4,940,838; U.S. Pat. No. 5,464,763) orparticle or microprojectile bombardment (U.S. Pat. No. 5,100,792; EP00444882B1; EP 00434616B1). Other plant transformation methods can alsobe used like microinjection (WO 09209696; WO 09400583A1; EP 175966B1),electroporation (EP00564595B1; EP00290395B1; WO 08706614A1) orPEG-mediated transformation of protoplasts etc. The choice of the methodfor vector delivery may depend on the plant species to be transformed.For example, microprojectile bombardment is generally preferred formonocot transformation, while for dicots, Agrobacterium-mediatedtransformation gives better results in general.

In the examples of the invention, we used transientAgrobacterium-mediated delivery of vectors (said heterologous DNA) intoNicotiana cells. However, said vectors may be stably introduced into theplants in accordance with any of the standard techniques suitable forstable or transient transformation of the plant species of interest.Transformation techniques for dicotyledonous are well known in the artand include Agrobacterium-based techniques and techniques which do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. Thesetechniques include PEG or electroporation mediated uptake, particlebombardment-mediated delivery and microinjection. Examples of thesetechniques are described in Paszkowski et al., EMBO J 3, 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199,169-177 (1985), Reich etal., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature327,70-73 (1987). In each case, the transformed cells are regenerated towhole plants using standard techniques.

Agrobacterium-mediated transformation is a preferred technique for thetransformation of dicotyledons because of its high transformationefficiency and its broad utility with many different species. The manycrop species which may be routinely transformed by Agrobacterium includetobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean,alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO87/07299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)).

In the examples of this invention, Agrobacterium-mediated delivery ofT-DNA for transient expression of gene(s) of interest (Vaquero et al.,1999, Proc. Natl. Acad. Sci. USA, 96, 11128-11133) was employed. Thismethod is an extremely useful tool not only for small-to-middle scalerecombinant protein production systems, but also for large-scaleexpression.

Release of viral replicon precursor stably incorporated into plantchromosomal DNA can be achieved using inducible or any other regulated(e.g. developmentally regulated) promoter. Inducible promoters can bedivided into two categories according to their induction conditions:those induced by abiotic factors (temperature, light, chemicalsubstances) and those that can be induced by biotic factors, forexample, pathogen or pest attack. Examples of the first category areheat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat.No. 5,847,102) promoters, a copper-inducible system (Mett et al., 1993,Proc. Natl. Acad. Sci., 90, 45674571), steroid-inducible systems (Aoyama& Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J.,14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-inducible system(Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and atetracycline-inducible system (Weinmann et al., 1994, Plant J., 5,559-569). One of the latest developments in the area of chemicallyinducible systems for plants is a chimaeric promoter that can beswitched on by glucocorticoid dexamethasone and switched off bytetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review onchemically inducible systems see: Zuo & Chua, (2000, Current Opin.Biotechnol., 11, 146-151) and Padidam, M (2003, Curr. Opin. Plant Biol.,6, 169-177). Other examples of inducible promoters are promoters, whichcontrol the expression of patogenesis-related (PR) genes in plants.These promoters can be induced by treatment of a plant with salicylicacid, an important component of plant signaling pathways in response topathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole orisonicotinic acid) which are capable of triggering PR gene expression(U.S. Pat. No. 5,942,662).

This invention is not limited to TMV and PVX-based vectors described inthe examples, but is applicable to replicons derived from other plantRNA viruses, subject to the establishment of expression systems derivedfrom said viral replicons. The best studied synergism in co-infectedplants is known for the PVX/PVY pair of viruses (Rochow & Ross, 1955,Virology, 1, 10-27; Goodman & Ross, 1974, Virology, 58, 16-24). It isvery likely that many of those viruses can co-replicate in the sameplant cell. Dietrich & Maiss (2003, J. Gen. Virol., 84, 2871-2876) haveshown that differently labelled pairs of viruses, e.g. plum pox virus(PPV) and potato virus X (PVX), tobacco vein mottling virus (TVMV) andPVX, Clover yellow vein virus (CIYVV) and PVX, can co-express differentreporter genes in the same infected region of plant tissue. Using thestrategy described in this invention, recombinant hetero-oligomericprotein expression systems for practically any pair of plant plus-sensesingle-stranded RNA virus-derived replicons that are capable ofco-replication in the same plant cell can be developed. For example,viral vectors based on alfalfa mosaic virus (AMV) of the genusalfamovirus can be used in this invention.

Genes of interest encoding for complex (hetero-oligomeric) proteins,their fragments (functional or non-functional) and their artificialderivatives and fusions can be expressed in plants or plants cells usingthe present invention. Many commercially valuable groups ofhetero-oligomeric proteins can be produced and purified using theinvention. Those groups include but not limited to industrial andresearch proteins as well as proteins for applications in the area ofhuman or animal health. However, the most preferred is the group ofimmune response proteins, specifically—monoclonal antibodies selectedfrom different classes of immunoglobulins (IgG, IgM, IgA and IgD) andtheir synthetic derivatives like mutant versions and different types offusions with other proteins or parts thereof.

EXAMPLES

The following examples are presented to illustrate the presentinvention. Modifications and variations may be made without departingfrom the spirit and scope of the invention.

Example 1 Lack of Co-Expression of GFP and DsRed from Two TMV-Based RNAViral Vectors that are Derived from the Same Plant RNA Virus

Leaf tissue was infected with two TMV-based viral vectors derived fromthe same plant RNA virus but containing two different genes of interest(FIG. 1). We tested this approach using the visual marker genes GFP andDsRed cloned into a TMV-based viral vector. The first construct,pICH17272 (see FIG. 1 of Marillonnet et al., 2005, Nat. Biotechnol., 23,718-723), contains GFP, and is similar to pICH18711 (see Internationalpatent application PCT/EP03/12530, published as WO2005049839;Marillonnet et al., 2005, Nat. Biotechnol., 23, 718-723) with theexception that the RdRP coding sequence in the vector contains 9 plantsintrons rather than 14. The pICH18722 (Marillonnet et al., 2005, Nat.Biotechnol., 23, 718-723) is built on the backbone of two closelyrelated strains of TMV, Cr-TMV (Dorokhov et al., 1994, FEBS Lett. 350,5-8) and TVCV (Lartey et al., 1994, Arch. Virol. 138, 287-298), andcontains the viral genome placed under control of a plant promoter, withthe coat protein sequence replaced by a heterologous sequence. Thepresence of 11 introns in the RdRP and MP increases the efficiency ofinitiation of viral replication after agrobacterium-mediated delivery ofthe vector to leaf tissue, and therefore increase the probability ofco-expression of the two viral vectors in the same cell. The pICH18505is similar to pICH17272 except that the GFP coding sequence has beenreplaced by the coding sequence of DsRed using Xho 1-Not 1 restrictionsites (FIG. 1). Detailed restriction maps of T-DNA regions of pICH18505and pICH17272 are shown in FIG. 1C. pICH17272 and pICH18505 weretransformed in Agrobacterium strain GV3101, and leaf tissue wasinfiltrated into Nicotiana benthamiana as previously described(Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857).Protoplasts were prepared from the infiltrated area 7 days postinfiltration (dpi) and observed under blue or red light under themicroscope. Very few protoplasts expressed both GFP and DsRed (FIG. 1A),even when protoplasts were prepared from the infiltrated area severaldays later. This suggests that once a cell becomes infected with a firstTMV-based viral vector, it becomes unable to be reinfected by a secondTMV-based vector.

Example 2 Co-Expression of GFP and DsRed from a Single Viral Replicon

Two genes of interest were expressed from an RNA viral vector, undercontrol of two separate subgenomic promoters (FIG. 2). Both subgenomicpromoters can be identical, but to avoid the risk of deletion betweenrepeated sequences in the construct during viral replication, it ispreferred to use subgenomic promoters from related TMV strains; in thiscase, the second subgenomic promoter and 3′ non-translated region comesfrom TMGMV strain U5 (Shivprasad et al, 1999, Virology, 255, 312-323;Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA 101, 6852-6857).Also, for convenience of cloning, GFP and DsRed were cloned in viralprovectors (described in WO02/088369 and by Marillonnet et al., 2004,Proc. Natl. Acad. Sci. USA, 101, 6852-6857), rather than in a completeassembled vector. The two provectors, pICH17388 and pICH15933 (FIG. 3),were converted to a fully functional TMV-based viral vector bysite-specific recombination between both provector modules in planta.pICH17388 is similar to pICHNOP (Marillonnet et al., 2004, Proc. Natl.Acad. Sci. USA, 101, 6852-6857) with the exception that 11 introns arepresent in viral sequences (as in pICH17272). pICH15933 is equivalent topICHGFPSYS (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA 101,6852-6857) except that the coding sequence of TMGMV U5 was replaced bythe coding sequence of DsRed.

The pICH15933 was transformed in Agrobacterium strain GV3101 andinfiltrated in Nicotiana benthamiana leaf together with pICH17388 andpICH10881 (FIG. 3). Five days after infiltration, all GFP-expressingfoci were also expressing DsRed showing excellent coexpression of twogenes in the same plant cells (FIG. 4).

Example 3 Expression of an Antibody from a Single Viral Replicon

The coding sequences of GFP and DsRed in pICH15933 were replaced by theIgG antibody light and heavy chains, respectively, resulting inconstruct pICH20241 (FIG. 5). As a control, the heavy and light chainswere cloned in a TMV-based provector, replacing the coding sequence ofpICH1740, resulting in constructs pICH20421 and pICH20431 (FIG. 5). ThepICH20241 was coinfiltrated in Nicotiana benthamiana leaves togetherwith pICH17388 and pICH10881 (FIG. 3).

Western blot analysis of total soluble protein extracted frominfiltrated leaves was performed with 1:6000 diluted anti-human IgGrabbit antibodies labelled with horseradish peroxidase (HRP) (Sigma).The results of the analysis are shown in FIG. 6 A. It is evident thatthe expression level of an antibody achieved with the help of a singleviral vector (lanes 6 & 7, FIG. 6) is significantly higher than thatachieved with the help of a strong constitutive promoter even in thepresence of the PTGS suppressor P19 (lanes 3-4, FIG. 6).

Example 4 Co-Expression of GFP and DsRed with TMV- and PVX-Based ViralVectors

An other strategy for coexpression of two genes is to use separate viralvectors built on different viruses that can coinfect and replicate inthe same cell. As an example, an expression vector based on potato virusX (PVX) can coexist in the same cell with TMV. Schematic representationsof two such non-competing viral vectors are shown in FIG. 7.

Inoculum of PVX (strain PV0014) was obtained from the German Collectionof Microorganisms and Cell Cultures (DSMZ) as infected dry leafmaterial, and was used for inoculation of Nicotiana bentamiana plants.Systemic leaves of inoculated plants that exhibited viral symptoms wereused for preparation of total RNA. 1^(st) strand cDNA was made usingprimers pvxpr2, pvxpr4 and pvxpr6. Three cDNA fragments were amplifiedby PCR from PVX cDNA using a Pfu-Taq polymerase mix:

Fragment 1 Amplified with Primers:

pvxpr1: (SEQ ID NO: 1) ttt ggtctc a tgaa gaaaactaaaccatacaccaccaacacaac Pvxpr2: (SEQ ID NO: 2) ctttttccagcccggagaccatttctgtgatgg 

Fragment 1 digested with BsaI.

Fragment 2 Amplified with Primers:

Pvxpr3: (SEQ ID NO: 3) ttt cgtctc a gggctggaaaaagaggacttccctgaagg Pvxpr4: (SEQ ID NO: 4) gagtcgtctcctgcataaacttgagcag 

Fragment 2 digested with Esp3I

Fragment 3 Amplified with Primers:

Pvx5: (SEQ ID NO: 5) ttt gaagac aa tgcaggagacgactccgcactgg Pvx6:(SEQ ID NO: 6) cg gacgtc tttttttttttttttttttttttt atttatattattcatacaatcaaaccagaaaatac

Fragment 3 digested with BpiI AatII

Fragment 4 containing Arabidopsis actin2 gene promoter, (An et al.,1996, Plant J., 10, 107-121) was amplified from Arabidopsis genomic DNAwith primers:

Act2pr1: (SEQ ID NO: 7) ttt acgcgt ttcgacaaaatttagaacgaacttaattatgAct2pr2: (SEQ ID NO: 8) ttt ggtctc a ttca ttcaaagcggagaggaaaatatatg

Fragment 4 Digested with Mlu1 and Bsa1.

As an alternative to the Arabidopsis Actin 2 promoter, the CaMV 35promoter was also used to drive transcription of PVX-based viral vector(see FIG. 10D, pICH20788; pICH21380). The general strategy of makingPVX-based vectors was used as described by Baulcombe and colleagues,(1995, Plant Journal, 7:1045-1053).

All four fragments were cloned together in a binary vector digested withMlu1 and Aatll. 20 clones of the resulting construct were transformed inAgrobacterium strain GV3101 and each clone infiltrated into one leaf ofa Nicotiana benthamiana plant. One week later, Nicotiana benthamianawere phenotypically screened for viral infection symptoms, and thepositive plasmid clones saved (pICHPVX).

A cloning vector was made from this complete functional cDNA for cloninga gene of interest downstream of the CP subgenomic promoter. The GFPgene was cloned as an example. An ATG in the CP subgenomic promoter areawas mutated to AGG (position 5651 in Genbank accession M95516). The GFPgene was cloned 3′ of the NheI site (coordinates 5662 to 5667). The3′end of PVX (coordinates 5618 to 6435) was cloned downstream of the GFPsequence, and this sequence was followed by a stretch of 12 to 24 A).This construct (pICH0130, FIG. 8) was agroinfiltrated into Nicotianabenthamiana leaf. GFP fluorescence appeared in the infiltrated area twoto three days after infiltration. Systemic movement of GFP appeared afew days later.

The pIC0130 was coinfiltrated into Nicotiana benthamiana leaf togetherwith vectors pICH17388, pICH10580 and pICH10881 (FIG. 3) providing forexpression of DsRed. Six days after co-infiltration, protoplasts wereprepared from the infiltrated area and observed under the microscopeunder blue light. Nearly all protoplasts expressed both GFP and dsRed,showing excellent level of coexpression FIG. 9).

Example 5 Expression of a Monoclonal Antibody with TMV and PVX-BasedVectors

The heavy and light chains of the IgG were cloned in a PVX vector,replacing the coding sequence of GFP in pIC0130, generating twoconstructs, pICH21240 and pICH21370, containing either the heavy or thelight chain, respectively (FIG. 10 A). Clones of TMV provector partscontaining either the light or the heavy chain of antibody were alsoconstructed (pICH20431 and pICH20421, FIG. 10A). Mixture of agrobacteriaproviding for two different chains expressed form PVX and TMV-basedvectors were infiltrated into N. benthamiana leaves and the expressionlevel of antibodies were measured by ELISA 10 days after inoculation.Results (FIG. 11) show an extremely high level of assembled functionalantibodies in case of co-expression of heavy and light chains from PVXand TMV-based vectors. These levels were significantly higher than thoseobtained from a TMV vector expressing both chains (EXAMPLE 3).

Yet another human tumor-specific monoclonal antibody belonging to IgG1subclass, anti-cancer mab, was subcloned into TMV and PVX-based vectorsas follows: The 730 NcoI-NotI fragment, containing the anti-cancer mablight chain coding sequence was blunted at NotI site and ligated intoTMV-based 3′ module cloning vector pICH11599 (FIG. 10B) opened withNcoI-XbaI and blunted at the XbaI site resulting in pICH21910 (FIG.10B). The same strategy was used to subclone the 1421 bp NcoI-NotIfragment with the anti-cancer mab heavy chain coding sequence intopICH11599 (FIG. 10B) that resulted in the pICH21920 (FIG. 10B)construct. PVX-based 3′ module for GFP expression pICH21282 (FIG. 10C)was created on the basis of TMV-based 3′ module cloning vector pICH10990(FIG. 10C). It was achieved in several cloning steps via insertion ofGFP coding sequence into pICH10990 (FIG. 10C) and consequent replacementof TMV 3′ untranslated region with 3′ untranslated region of PVX.Similarly, TMV-based 3′ pro-vector module pICH21920 (FIG. 10B) wasconverted into PVX-based 3′ pro-vector construct pICH22250 (FIG. 10C) byreplacement of TMV 3′ untranslated region with 3′ untranslated region ofPVX. It was achieved by ligation of the 575 bp HindIII-NdeI fragment ofpICH21282 (FIG. 10C) blunted at the HindIII site with SalI-NdeI fragmentof pICH21920 blunted at the SalI site. A 723 bp NcoI-EcoRI fragmentcontaining coding sequence of anti-cancer mab light chain was subclonedinto pICH22250 (FIG. 10C) digested with NcoI-EcoRI, resulting inpICH22240 (FIG. 10C).

The 5′ provector pICH21380 (FIG. 10D) was made in the following way:construct pICH17388 (FIG. 3B) was used as template to PCR amplify thefragment with the help of primers pv5p5F (SEQ ID NO: 9) (5′ cagctagcaacaaacaagaa aggtaagttt c-3′) and pv5p5R (SEQ ID NO: 10) (5′-tctgagctctgcatgctacg cccccaactg agag-3′), digested with Nhe1 and Sac1 restrictionenzymes and ligated with large Nhe1-Sac1 fragment of pICH20788,replacing 3′ part of PVX vector with 5′ end of intron and AttPrecombination site. The scheme of cloning is shown in FIG. 10D. Thestrategy of using 5′ provectors with 3′ provectors in order to assemblea viral vector in planta via site-specific recombination was asdescribed earlier (Marillonnet et a!, 2004, Proc. Nat!. Acad. Sci. USA,101:6852-6857).

Agroinfiltration Procedure

All resulting constructs were transformed in Agrobacterium strain GV3101using a standard electroporation protocol and used for furtheragroinfiltration of Nicotiana benthamiana leaves. Equal volumes ofovernight grown Agrobacterium cultures, OD600 ranging from 1.8 to 2.5,were mixed and sedimented at 6000 g for 3 min. The pellet wasresuspended in a solution containing 10 mM MES (pH5.5) and 10 mM MgSO4,resulting in 10⁻¹ dilution for each individual culture. Leaves of 6-8week-old greenhouse-grown Nicotiana benthamiana plants were infiltratedby using a syringe without needle. In the case of using provectors foragroinfiltration, the agrobacterial mix usually carried 5 differentagrobacterial strains each of them providing for one of the followingcomponents: recombinase (integrase) source (pICH10881); 5′PVX provector(pICH21380); one 3′ PVX provector providing for one of the IgG chains(either pICH22250 for heavy chain or pICH22240 for light chain,depending on which complementary chain provided by TMV provector); 5′TMV provector (pICH17388); one 3′ TMV provector (either pICH21910encoding for light chain or pICH21920 encoding for heavy chain).

SDS-PAGE and Western Blot

100 mg samples of N. benthamiana leaves infiltrated with Agrobacteriumwere ground in liquid nitrogen with 300p1 protein extraction buffer(0.1M Tris, 150 mM NaCl and 0.1% Tween 20, pH8.0). Crude leaf extractswere resolved on 10% (non-reducing conditions) or 12% (reducingconditions) polyacrylamide gels using the buffer system of Laemmlifollowed by Coomassie Brilliant Blue staining. For Western Blotanalysis, proteins from SDS-PAGE were electrophoretically transferred toa Hybond P membrane (Amersham Biosciences, UK), using Blotting Buffer(25 mM Tris, 192 mM glycin, 20% methanol, pH8.3). Membranes were blockedwith 5% skim milk in TBST (50 mM Tris, 100 mM NaCl, 0.05% Tween 20,pH7.4) and probed with goat anti-human lambda light chain HRP-conjugatedantibodies (Sigma, UK) or goat anti-human IgG gamma chain specificHRP-conjugated antibodies (Sigma, UK) diluted in 2.5% skim milk/TBS1:10000 and 1:5000, respectively. Bound antibodies were detected usingECL Detection Reagent (Amersham Biosciences, UK). The results ofelectrophoretic separations and Western blot analyses are shown in FIG.11B-11D. Comparison with IgG standard showed that the yield ofmonoclonal antibodies in plant tissue reaches 0.2-0.35 mg per gram offresh leaf biomass.

Isolation of Plant-Derived Recombinant Mabs

Total soluble proteins were extracted from agroinoculated Nicotianabenthamiana leaf areas with buffer containing 100 mM sodium phosphate,150 mM NaCl and 0.05% Tween 20 (pH8.0). Small-scale isolation ofanti-cancer mab from crude protein extracts was performed using one-stepaffinity purification with Protein A Magnetic Beads (New EnglandBiolabs, USA) according to the instructions of manufacturer.Coomassie-stained gel with purified plant-derived Mab is shown in FIG.11B.

Example 6 Expression of Follicule Stimulating Hormone (FSH) with TMV andPVX-Based Vectors

The genes (cDNA) encoding for alpha (nucleotides corresponding to thecoding sequences 101-463 of GenBank Acc. No. NM_(—)173901) and beta(nucleotides corresponding to the coding sequences 70-459 of GenBankAcc. No. NM_(—)174060) polypeptides of bovine FSH were synthesizedflanked with restriction sites (5′ Nco1—3′EcoR1 for FSH-alpha andFSH-beta subunits) and cloned in TMV pro-vector (pICH20431, derivativeof pICH11599, FIG. 10B) and PVX vector (pICH21240), replacing the codingsequences for the heavy and light chains of IgG and generating twoconstructs, pICH-FSHA and pICH-FSHB, containing the sequences encodingfor subunit alpha and subunit beta, respectively (FIG. 12). Mixture ofagrobacteria providing for two different subunits expressed form PVX andTMV-based vectors were prepared and infiltrated into N. benthamianaleaves as described above and the expression level of heterodimeric FSHwas measured by ELISA 10 days after inoculation using commerciallyavailable FSH dimer-specific (FSH117) antibodies and detected withTropix chemiluminescent system (Tropix, Bedford, Mass.).

Example 7 Expression of IgM with TMV and PVX-Based Vectors

The coding sequences of GFP and DsRed in pICH15933 were replaced by thesequences encoding IgM light and J chains respectively, resulting inconstruct pICH-MLCJ (FIG. 13). The gene encoding for IgM heavy chain wascloned in PVX vector (pICH21240), replacing the coding sequences for IgGheavy chain and generating construct pICH-MHC (FIG. 13). Mixture ofagrobacteria providing for three different chains of IgM expressed formPVX and TMV-based vectors were infiltrated into N. benthamiana leavesand the expression level of hetero-oligomeric complex was measured byELISA 10 days after inoculation using commercially availableIgM-specific antibodies.

The contents of European patent application No. 05 001 819.1 and of U.S.patent application No. 60/593,606, the priorities of which are claimedby the present patent application, are incorporated herein by referencein their entireties.

1-42. (canceled)
 43. A process of producing in a plant, in plant tissue,or in plant cells a hetero-oligomeric protein comprising at least afirst and a second protein subunit, said process comprising expressingin plant cells at least said first and said second protein subunit byproviding to said plant, said plant tissue or said plant cells a firstand a second plus-sense single-stranded RNA viral vector, said firstviral vector encoding at least said first protein subunit, said secondviral vector encoding at least said second protein subunit, whereby saidfirst viral vector and said second viral vector are (a) non-competingviral vectors; (b) non-competing viral vectors in that they are notderived from viruses of the same virus species; (c) non-competing viralvectors in that they differ in sequence portions other than sequenceportions coding for said first and said second protein subunits; (d)non-competing viral vectors in that the replicase ORF(s) of said firstviral vector and the replicase ORF(s) of said second viral vector have ahomology of at most 90%; or (e) non-competing viral vectors in that thereplicase ORF(s) of said first viral vector and the replicase ORF(s) ofsaid second viral vector have an identity of at most 85%.
 44. Theprocess according to claim 43, followed by isolating saidhetero-oligomeric protein from said plant, said plant tissue, or saidplant cells.
 45. The process according to claim 43, wherein said firstviral vector contains a first heterologous sequence encoding said firstprotein subunit and said second viral vector contains a secondheterologous sequence encoding said second protein subunit, wherebyexpression of said first and/or of said second protein subunit is underthe control of a sub-genomic promoter.
 46. The process according toclaim 43, wherein said first viral vector and/or said second viralvector is/are devoid of a functional coat protein ORF, a functionalmovement protein ORF, and/or a functional origin of viral particleassembly.
 47. The process according to claim 43, wherein said firstviral vector is derived from a virus belonging to the genus Potexvirusand said second viral vector is derived from a virus belonging to thegenus Potyvirus.
 48. The process according to claim 47, wherein saidfirst viral vector is derived from Potato Virus X and said second viralvector is derived from Potato Virus Y.
 49. The process according toclaim 43, wherein said first viral vector is derived from a virusbelonging to the genus Potexvirus and said second viral vector isderived from a virus belonging to the genus Tobamovirus.
 50. The processaccording to claim 49, wherein said first viral vector is derived fromPotato Virus X and said second viral vector is derived from TobaccoMosaic Virus.
 51. The process according to claim 43, wherein said firstand said second viral vectors have at most 60% sequence homology. 52.The process according to claim 43, wherein said hetero-oligomericprotein is an antibody.
 53. The process according to claim 43, whereinsaid viral vector(s) is (are) transiently provided to said plant, planttissue, or plant cells.
 54. The process according to claim 53, whereinsaid viral vector(s) is (are) transiently provided to said plant, planttissue, or plant cells by Agrobacterium transfection.
 55. The processaccording to claim 43, wherein said RNA viral vector(s) is (are) stablyincorporated into plant chromosomal DNA as DNA precursors of said RNAviral vectors.
 56. The process according to claim 55, wherein controlledrelease of said RNA viral vector(s) from said DNA precursors is (are)provided by inducible promoters.
 57. The process according to claim 52,wherein said antibody is an immunoglobulin comprising at least a portionof an antigen binding domain.
 58. The method according to claim 52,wherein said antibody or its derivative belongs to the immunoglobulin Gclass, to the immunoglobulin A class, to the immunoglobulin M class, tothe immunoglobulin D class, or to the immunoglobulin E class.
 59. Theprocess according to claim 43, wherein said hetero-oligomeric protein isinsulin.
 60. The process according to claim 43, wherein at least one orat least two or more subunits of said hetero-oligomeric proteincontain(s) an endoplasmatic reticulum retention signal KDEL.
 61. Theprocess according to claim 43, wherein said plant is a monocot or adicot plant.
 62. The process according to claim 61, wherein said dicotplant belongs to the Solanacea family, Brassicacea family, the Legumefamily, or to the family Chenopodiaceae.